Potential Activities of Centella asiatica Leaf Extract against Pathogenic Bacteria-Associated Biofilms and Its Anti-Inflammatory Effects

The medicinal value of Centella asiatica leaf extract was evaluated as an alternative treatment. The chemical composition of the leaf extract was analyzed, and the biological activities were determined. High-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) was used to identify the asiatic acid, madasiatic acid, and madecassic acid/Brahmic acid isolated from the ethanolic extract. The plant extract at 25 mg/disk was found to inhibit both Gram-positive and Gram-negative pathogenic bacteria by the agar disk diffusion test. The MIC and MBC of the ethanolic extracts were better than those of the aqueous extracts. The ethanolic extracts showed antibacterial activity against Gram-positive bacteria with MICs and MBCs ranging from 1.024 to 2.048 mg/mL and 2.048 to 4.096 mg/mL, respectively. The remarkable antibacterial activities were observed against S. mutans. The ethanolic extract at a concentration of 1/2 × MIC exhibited the inhibition effect on S. mutans biofilm formation like the activity of 0.2% chlorhexidine and significantly modified hydrophobicity of the bacterial cell surface. The effects were confirmed via molecular docking analysis. The binding affinities of asiatic acid, madecassic acid, and madasiatic acid with glucosyltransferase C (GtfC) of S. mutans exhibited superior strength in comparison with alpha-acarbose and chlorhexidine. Moreover, the nitric oxide (NO) secretion of RAW247.6 cells was determined after treating the cells with concentrations of the extract. The C. asiatica ethanolic extract can inhibit the secretion of NO, which can inhibit the inflammatory process. The findings indicate the applications of the C. asiatica ethanolic extract as the alternative anti-S. mutans agent and could be used for further formulation for the treatment and prevention of dental diseases and inflammatory injury in the oral cavity.


Introduction
Centella asiatica, commonly known as Gotu kola, boasts a rich historical signifcance intertwined with various cultures and traditions.Its roots in traditional medicine stretch back centuries, with ancient texts from Ayurveda, traditional Chinese medicine (TCM), and traditional African medicine all lauding its therapeutic properties.In Ayurveda, this herbal plant has been revered as a "Medhya Rasayana," an herb that promotes mental clarity and cognitive function.It has been used to enhance memory, improve concentration, and alleviate anxiety and stress.An aqueous leaf extract of the plant has been shown to increase synaptic density, improve cognitive function, and demonstrate promising efects on memory retention in rats.Researchers attributed these efects to the herb's ability to enhance the expression of the antioxidant response gene NRF2 [1].Moreover, C. asiatica has garnered attention for its neuroprotective efects, with research suggesting its potential to mitigate agerelated cognitive decline and neurodegenerative disorders, such as Alzheimer's disease [2].Gray et al. investigated the neuroprotective potential of C. asiatica in an animal model of Alzheimer's disease.Te fndings suggested that the herb's anti-infammatory and antioxidant properties may help protect against neurodegeneration and cognitive decline [3].In TCM, C. asiatica is valued for its ability to cool infammation, promote wound healing, and support the health of the skin.It is often utilized to treat conditions, such as eczema, psoriasis, and various infammatory skin disorders.Somboonwong et al. demonstrated the anti-infammatory efects of the plant extract and its ability to accelerate wound healing.Te study suggested that the herb promotes collagen synthesis and angiogenesis, contributing to faster tissue repair [4].Furthermore, emerging research suggests that C. asiatica may have benefts in managing conditions, such as diabetes, cardiovascular diseases, and gastrointestinal disorder, although further clinical studies are warranted to elucidate its full therapeutic potential in these areas.Te efects of the plant extract on cardiovascular health and metabolic disorders were explored.Te study indicated that C. asiatica supplementation may have benefcial efects on lipid metabolism, blood pressure regulation, and insulin sensitivity [5].
Te traditional use of C. asiatica has gained scientifc interest due to its diverse bioactive components, such as triterpenoids, favonoids, and asiaticoside [6,7].Tese compounds have been extensively studied for their pharmacological activities, including anti-infammatory, antioxidant, and neuroprotective efects [2,6].Tese properties provide a scientifc basis for the traditional use of C. asiatica across diferent medical conditions.
Several studies have reported the antibacterial potential of C. asiatica extracts and their bioactive compounds.Research conducted by Pitinidhipat et al. demonstrated the inhibitory efects of C. asiatica against both Gram-positive and Gram-negative bacteria [8].Te essential oil extracts from C. asiatica demonstrated the activity to inhibit Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei [9].Te study highlighted the presence of bioactive compounds, such as triterpenoids and favonoids, which contribute to the antibacterial activity [10,11].Tis fnding suggests that the plant extract may have the potential to serve as a natural alternative to conventional antibiotics.It has a high content of antioxidant compounds, including asiaticoside and madecassoside [12,13].A study by Chintapanti et al. explored the antioxidant properties of C. asiatica in an animal model, demonstrating its ability to enhance antioxidant enzymes and decrease oxidative stress markers [14].Tese fndings indicated that the plant may have a role in mitigating oxidative damage associated with various diseases including cell injuries caused by the infections.
Moreover, infammation underlies many chronic diseases, making the search for natural anti-infammatory agents crucial.A study by Dong et al. investigated the anti-infammatory efects of C. asiatica in a mouse model of infammation-induced lung injury.Te researchers found that treatment with the herbal extract signifcantly reduced infammation mediators and improved lung function [15].Te mentioned studies highlight the bioactive compounds present in C. asiatica and their pharmacological activities, ofering insights into its potential therapeutic applications.However, further research is required to elucidate the mechanisms of action and optimize the usage of C. asiatica in clinical practice.Understanding the efciency of C. asiatica in various medical applications could not only contribute to the development of new natural remedies but also help in the discovery of novel bioactive compounds for pharmaceutical applications.Hence, this research article aims to consolidate the current knowledge and encourage further investigations into the vast potential of C. asiatica as a valuable medicinal plant.

Plant Materials and Preparation of Herbal Extracts.
Fresh Centella asiatica leaves were collected in 2021 from Chian Yai Subdistrict and Mae Chao Yu Hua Subdistrict, Nakhon Si Tammarat Province, Tailand.Herbarium voucher specimens were Centella asiatica SM 0324030901.Tey were deposited at Applied Tai Traditional Medicine, School of Medicine, Walailak University, Nakhon Si Tammarat, Tailand.Te plant materials were dried using a hot air oven at 60 °C for 72 hours.All dried herbs were ground into coarse powders.To obtain crude ethanolic extracts, pulverized herbs were macerated in 95% ethanol (1 : 10 w/v) for 7 days.For aqueous extracts, the herbs were subjected to 6-hour cycles of Soxhlet extraction.Four repeated cycles were performed using a sample-to-solvent ratio (g/mL) of 1 : 10 at the temperature of 45 °± 2 °C.Te macerates were then fltered and dried using a rotary evaporator.Te crude extracts were stored at −20 °C until further use.Te plant extracts were dissolved in dimethyl sulfoxide (DMSO) and diluted to obtain a fnal concentration of the solvent less than 10% before use for antibacterial assay and antibioflm formation testing.

Qualitative Phytochemical Analysis of Herbal Extracts.
Te herbal extract and triterpene standards were prepared at 1.0 mg/mL in methanol and fltered through a 0.45 μm nylon membrane syringe flter and subjected to high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA).Te analysis was carried out with

Microbial Adhesion to Hydrocarbon (MATH) Test.
MATH assay was performed to evaluate the bacterial cell surface hydrophobicity.S. mutans ATCC 25175 was used as a representative of bioflm-producing bacteria in this assay.Te bacteria were cultured in BHI broth containing the plant ethanolic extract with the fnal concentration of 1/4 × MIC to 4 × MIC, at 37 °C for 4 hours.Te cell pellets were collected by centrifugation at 4000 × g for 5 min and washed twice with sterile saline solution.Te cell density was adjusted to an OD of 0.3 at 600 nm (OD initial).Te bacterial cells incubated without the extract were used as a control.Tree milliliters of the cell suspension were put into a glass tube adding 0.25 mL of toluene reagent.Te tubes were thoroughly mixed for 2-3 minutes using a vortex machine and kept at room temperature for 10 min.After the toluene phase had separated from the culture phase, the OD of the aqueous phase (OD fnal) was determined at 600 nm by spectrophotometry.Te bacteria with a hydrophobic index greater than 60% were classifed as hydrophobic.Te tests were performed in triplicate.Te hydrophobicity index (%) was calculated as follows: Tese structures were subjected to geometry optimization and energy minimization using the Merck molecular force feld (MMFF94s) [18,19] in the Avogadro software version 1.2.0.Gasteiger charges were added, and nonpolar hydrogen atoms were merged using AutoDockTools v. 4.2.6.Te resulting ligand structures were saved in the protein data bank, partial charge, and atom type (PDBQT) format.AutoDockTools was used for the preparation of the protein structure, which involved the removal of cocrystallized ligands (alpha-acarbose and 2-(n-morpholino)-ethanesulfonic acid), the removal of water molecules, the addition of polar hydrogens, and the assignment of Kollman charges.Te active site of GtfC, including Glu515, Asp477, Asp588, Arg475, His587, Tyr916, Tyr430, Leu433, Asn481, and Trp517, was determined based on the amino acids interacting with alpha-acarbose present in the protein.A grid box was created to cover these regions using AutoDockTools, with dimensions of 24 × 24 × 24 Å3 for x, y, and z points, a grid spacing of 1.000 Å, and the center coordinates (x � 192.011, y � 45.529, z � 194.613Å).Molecular docking was performed using AutoDock Vina v. 1.1.2,with an exhaustiveness value of 24 and other parameters set as default.
Te compounds with the lowest binding energy (kcal/mol) and minimum root mean square deviation (RMSD) were selected as the most suitable docking poses.To validate the docking process, the native ligand (alpha-acarbose) molecule was redocked into the identifed active site of GtfC under the same conditions, and the resulting RMSD of the redocked ligand should be below 2.5 Å to confrm the reliability of this method before proceeding with the experiment.Te hydrogen bonds and hydrophobic interactions between ligand atoms and amino acid residues of GtfC were identifed using the Protein-Ligand Interaction Profler (PLIP) online web server with default parameters.Te protein-ligand complexes were visualized using the PyMOL molecular graphics system v. 2.5.2.Te tested bacterial stains were all susceptible to standard antibiotics according to CLSI antibacterial testing standards.

Phytochemical Components of the Ethanolic Extracts of C. asiatica.
Te ethanolic extracts of C. asiatica demonstrated stronger antibacterial efects compared to the aqueous extracts.Consequently, a qualitative analysis of the ethanolic extracts was conducted using high-performance liquid chromatography coupled with a photodiode array (HPLC-PDA).Te analysis was performed using both negative and positive ionization modes to determine the chemical composition of the extract.Drawing from prior research, we identifed candidate compounds in the extract that might be responsible for its antibacterial properties.Te proposed negative and positive ions corresponding to various compounds are presented in Table 3. Tree principal compounds of madecassic acid, madasiatic acid, and asiatic acid were detected in the ethanolic extracts of C. asiatica leaves at specifc retention times (RTs).Madecassic acid was observed at an RT of 6.7, madasiatic acid at 7.3, and asiatic acid at 7.7.Furthermore, the HPLC-PDA chromatogram of triterpene acid standards, along with the isolated compounds, is illustrated in Figure 1.Te photodiode array detector was used to measure light absorption across a broad range of wavelengths, capturing a spectral profle for each peak of the compounds (Figure 1(b)), which was then compared with the spectra of the reference standards (Figure 1(a)).

Efects of C. asiatica Ethanolic Extracts on S. mutans Cell
Surface Hydrophobicity.Te modifcation efects of the ethanolic extracts on bacterial cell surface were determined.We hypothesized that the plant extract may modify the cell surface hydrophobic properties, which afects the aggregation activity of the bacteria to the host cells.S. mutans ATCC 25175 were selected as the representatives of hyper-bioflmproducing bacterial strains in this experiment.Te bacteria were classifed as hydrophobic bacteria with a hydrophobicity index greater than 60%.After treating the bacterial cells with the ethanolic extracts of C. asiatica at a concentration of 1/8 × MIC, 1/4 × MIC, 1/2 × MIC, and the MIC, the S. mutans cells possessed signifcant lower level of hydrophobicity than the untreated cells.Te ethanolic extract modifed the bacterial cell surface by decreasing the hydrophobicity in a concentration-dependent manner.Te concentration of 1/4 × MIC of the ethanolic extracts could signifcantly reduce the cell hydrophobicity of S. mutans, which is shown in Figure 3. Te hydrophobicity of the bacterial cells was signifcantly decreased to reach an index lower than 50% by exposure to the concentration of 1/ 2 × MIC of the ethanolic extracts.

Molecular Docking Analysis.
Te results obtained from the molecular docking analysis in this study revealed the binding afnities of GtfC with several compounds, including alpha-acarbose (a cocrystallized ligand and known Advances in Pharmacological and Pharmaceutical Sciences inhibitor), chlorhexidine (employed as a positive control), and natural compounds derived from C. asiatica, namely, asiatic acid, madecassic acid, and madasiatic acid.Te computed binding afnities were determined to be −8.2 kcal/ mol, −8.4 kcal/mol, −8.8 kcal/mol, −10.0 kcal/mol, and −10.0 kcal/mol, respectively (Table 4).It is important to note that in the context of molecular docking analysis, higher negative scores correspond to stronger binding afnities between the ligand and the protein [20].Remarkably, in terms of energy considerations, the binding afnities of asiatic acid, madecassic acid, and madasiatic acid with GtfC exhibited superior strength in comparison with alphaacarbose (a known inhibitor) and chlorhexidine (a positive control) when interacting with GtfC.

Discussion
C. asiatica has been revered for its medicinal properties in traditional medicine systems for centuries.It originates from Southeast Asia [6,7] and belongs to the Apiaceae family.Scientifc research has begun to validate many of these traditional uses, shedding light on the diverse medicinal benefts of this plant [21][22][23].One of the notable medicinal benefts of C. asiatica is its potential as an anti-infammatory agent.Studies have shown that it can help reduce infammation in the body and alleviate symptoms associated with conditions [15,24].C. asiatica also exhibits antioxidant properties, which can help protect the body against oxidative stress and damage caused by harmful free radicals [6].Furthermore, C. asiatica has demonstrated abilities to support the nervous system.It is believed to have a positive impact on cognitive function, memory, and mental clarity [2].Some studies have suggested that it may even have potential in the management of neurological disorders, such as Alzheimer's disease.Additionally, the plant has shown promise in its antibacterial properties.It has been found to inhibit the growth of various harmful bacteria, including strains that are resistant to antibiotics [8,[25][26][27].Tis makes it a potentially valuable natural alternative for combating bacterial infections.Moreover, it has been associated with potential benefts for wound healing and scar reduction.It is believed to stimulate production and enhance skin cell regeneration, aiding in the recovery of wounds and preventing excessive scarring.It is important to note that while C. asiatica shows promising medicinal benefts, further research is still needed to fully understand its mechanisms of action and confrm its efcacy.As with any herbal remedy, it is recommended to consult with a healthcare professional before incorporating C. asiatica into your medical treatment.
Te ethanolic extracts exhibited superior antibacterial activity in comparison with the aqueous extracts.Tis was evidenced by the lower MIC and MBC values observed for the ethanolic extract.Tese fndings indicate the efcacy of the ethanolic extract at lower concentrations, suggesting its potential as a powerful antibacterial agent.In contrast, the aqueous extracts demonstrated higher MIC and MBC values, implying that higher concentrations are necessary to achieve similar antibacterial efects.Tis emphasizes the signifcance of the solvent used in the extraction process, specifcally in the context of the agar disk difusion method.
Furthermore, our investigation revealed that the C. asiatica ethanolic extract displayed notable inhibitory efects primarily against Gram-positive strains, whereas its impact on Gram-negative strains was relatively weaker.Tis distinction was evident from the larger inhibition zones observed for Gram-positive bacteria (ranging from 9.6 to 14.3 mm), indicating a potentially robust inhibitory efect.In contrast, the inhibition zones observed for Gram-negative bacteria were generally smaller (ranging from 7.3 to 9.2 mm), implying a comparatively weaker efect against this bacterial group.Te results were like the study conducted by Soyingbe et al. demonstrated the ability of C. asiatica extract to inhibit both Gram-positive and Gram-negative bacterial strains [27].However, our fndings specifcally highlight the substantial inhibitory efect of the ethanolic extract against S. mutans ATCC 25175, as evidenced by an inhibition zone of 14.3 mm.Remarkably, this outcome was like the inhibitory efects achieved by a vancomycin drug.Te relevance of this fnding lies in its implications for oral health, as S. mutans is an important contributor to dental caries and other oral diseases [28,29].Additionally, it is worth noting that the choice of solvent used in the extraction process signifcantly infuences the antibacterial activity of the extracts [30].
While our current fndings ofer valuable insights into the antibacterial potential of the C. asiatica ethanolic extracts, further study is necessary to advance our understanding in this area.Additionally, it is crucial to thoroughly evaluate the safety and efcacy of the C. asiatica

8
Advances in Pharmacological and Pharmaceutical Sciences Two interaction with amino acid residues.Amino acid residues in the active site of GtfC of S. mutans, as shown in bold.
Advances in Pharmacological and Pharmaceutical Sciences extracts for various therapeutic applications, particularly in the context of mammalian cells and bacterial infection management.
Te investigations focus on the isolation and characterization of the bioactive compounds responsible for the observed efects.HPLC-PDA was used to analyze the ethanolic extracts of C. asiatica and contributed signifcant insights into the chemical composition of these extracts.Tis analytical technique enabled the identifcation of compounds likely responsible for the observed antibacterial activity.Among the numerous compounds detected in the ethanolic extracts, three principal compounds were identifed, such as madecassic acid, madasiatic acid, and asiatic acid [2,30].Asiatic acid is a triterpene compound with a chemical structure of "3,3′-methylene-bis(4-hydroxybenzaldehyde)," which is prominently found in C. asiatica [15,24].Extensive research has associated asiatic acid with various bioactive properties, including antibacterial activity [7,31].Similarly, madecassic acid, a terpene compound, presents in the ethanolic extracts of C. asiatica leaves [12].It has also been linked to diverse biological activities [7,12,32], including potential antibacterial efects.Te present study revealed the presence of madasiatic acid, another bioactive compound detected at a retention time of 7.3 minutes, sharing similarities with asiatic acid.Tis compound has been reported to exhibit wound healing and neuroprotective efects [6,21].Te antibacterial activity observed with the ethanolic extracts can likely be attributed to the presence of these bioactive compounds, warranting further investigation into their contributions and potential therapeutic applications.
Exploring the detailed mechanisms underlying the antibacterial efects of these compounds could lead to the development of targeted antibacterial agents or pharmaceutical applications derived from C. asiatica.Continued academic and scientifc research in this area holds great promise for advancements in antibacterial therapy and the feld of natural product pharmacology.Such dental caries, a prevalent oral disease, is primarily attributed to the formation of bioflms, particularly by the bacterium S. mutans [26,28].Tis bacterium plays a pivotal role in the development of dental cavities, possessing cariogenic properties  Advances in Pharmacological and Pharmaceutical Sciences [29,33].Moreover, this pathogen holds signifcant relevance to cardiovascular infammation, as it can potentially enter the bloodstream through infamed gums or periodontal pockets, leading to life-threatening complications [34].In light of these factors, our study investigated the inhibitory efects of the C. asiatica ethanolic extracts on the bioflm production of S. mutans ATCC 25175 as a representative strain, which is closely associated with dental diseases.Tis study demonstrated a signifcant concentrationdependent inhibition of bioflm production by the ethanolic extracts.Notably, noteworthy inhibition of bioflm formation was observed at concentrations of sub-MIC at 1/ 4 × MIC and 1/2 × MIC of the extracts.Interestingly, the ethanolic extract at 1/2 × MIC exhibited levels of inhibitory efects comparable to that of the standard drug 0.2% chlorhexidine.Te results suggest that the ethanolic extracts possess the potential to serve as efective inhibitors of S. mutans bioflm formation.Moreover, we hypothesized that the ethanolic extracts might modify the surface properties of S. mutans cell surface, potentially afecting their aggregation behavior and bioflm formation [28,35].Te MATH assay demonstrated that the ethanolic extracts indeed induced signifcant modifcations in the hydrophobicity of the bacterial cells.After exposure to concentrations at sub-MIC of the ethanolic extracts, the hydrophobicity of the bacterial cells was signifcantly decreased.Tis observation is crucial as changes in cell surface hydrophobicity can impact the bacterial aggregation and adhesion virulent factors, which are key processes in bioflm formation [35].Te ability of the ethanolic extracts to reduce the hydrophobicity of S. mutans cells suggests a mechanism through which these extracts inhibit bioflm formation [29].
Previous studies have extensively elucidated the mechanism by which GtfC catalyzes glucan formation [36].Tis process involves the hydrolysis of sucrose, a natural substrate for GtfC, through proton attack.Te resulting glycosyl moiety is bound to amino acid residues in subsite-1 of GtfC as an intermediate, while fructose is released from subsite+1 of the enzyme.Critical amino acid residues, namely, Arg475, Asp477, Glu515, His587, Asp588, and Tyr916, play a pivotal role in subsite-1, thereby facilitating the synthesis of glucans.Similarly, amino acid residues Tyr430, Leu433, and Trp517, located at subsite+1, are responsible for recognizing the glucosyl moiety [17,37].Furthermore, previous studies have consistently confrmed the pivotal role of Asp588, Tyr517, and Asn481 in catalyzing the hydrolysis of sucrose, which acts as the natural substrate for GtfC.Importantly, any favorable interaction, particularly involving hydrogen bonding, signifcantly disrupts the native catalytic functions of these residues, ultimately leading to the inhibition of the enzyme [17,38].Te molecular docking analysis of natural compounds isolated from C. asiatica, specifcally asiatic acid, madecassic acid, and madasiatic acid, revealed their interactions with multiple amino acid residues in the catalytic site of GtfC, involving hydrogen bonding and hydrophobic interactions.Tese interactions may impede the utilization of the natural substrate (sucrose) for water-insoluble glucan synthesis.Consequently, asiatic acid, madecassic acid, and madasiatic acid hold signifcant potential as lead compounds for the development of preventive agents against bioflm formation caused by S. mutans.
Moreover, these fndings demonstrate the efect of ethanolic extract of C. asiatica on NO production in LPSstimulated RAW 264.7 cells.C. asiatica ethanol extract signifcantly reduced NO production.Tis reduction is noteworthy, as high NO levels are often associated with infammation and oxidative stress [39].Interestingly, the value of NO production in aspirin-treated LPS-stimulated RAW 264.7 cells was not signifcantly diferent from that of C. asiatica ethanol extract.Tis suggests that C. asiatica ethanol extract was as efective as aspirin in reducing NO levels in these stimulated cells.Tese results are promising and indicate that C. asiatica ethanol extract has the potential to reduce infammation, which corresponds to the antiinfammatory of madecassic acid [32,40] and asiatic acid, the bioactive compounds [13,15,24,41].

Conclusion
Te study suggested that the ethanolic extracts of C. asiatica could be used as natural agents against bacterial infections especially S. mutans infections, as they exhibited promising antibacterial activities.Te modifcations of bacterial cell surface were observed after treating the bacterial cells with the ethanolic extracts by decreasing the cell surface hydrophobicity and signifcantly reducing bacterial bioflm formation within 24 hours.Te inhibitory actions on bacterial bioflms were confrmed by a demonstration of 3D structural interactions between the compounds isolated Advances in Pharmacological and Pharmaceutical Sciences from the C. asiatic ethanolic extract and the GtfB amino acids of S. mutans.Te molecular docking revealed a high docking score of asiatic acid, madecassic acid, and madasiatic acid isolated from the plant ethanolic extract.Te activity was close to those of chlorhexidine, suggesting that it was an in vitro bioflm inhibitor in this study.Moreover, the ethanolic extract demonstrated signifcant reducing NO production in LPS-stimulated RAW 264.7 cells.Te antiinfammatory activity could be compared to the action of the aspirin drug.Te fndings indicated the applications of the C. asiatica ethanolic extract as the alternative anti-S.mutans agent and could be used for further formulation for the treatment and prevention of dental diseases and infammatory injury in the oral cavity.
Cytotoxicity.Cytotoxicity of the ethanolic C. asiatica extracts in RAW 264.7 cells was performed using MTT assays.Te dose-response/sigmoidal curve ftting analysis of percent cell viability was established.LC50 and LC10 of ethanolic C. asiatica extracts were

Figure 1 :
Figure 1: High-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) chromatograms of triterpene acid mixed standards (a) and Centella asiatica ethanolic extract (b) were obtained, along with the corresponding chemical structures of the isolated compounds (c).Te spectra exhibited distinct peaks corresponding to madecassic acid (1), madasiatic acid (2), and asiatic acid (3).

Figure 2 :Figure 3 :
Figure 2: Inhibitory efects of Centella asiatica ethanolic extract at the concentration of 1/4 × MIC, 1/2 × MIC, and the MIC on S. mutans bioflm formation within 24 hours compared to the activities of 0.2% chlorhexidine standard solution.

Figure 4 :
Figure 4: Docking poses of alpha-acarbose (a cocrystallized ligand and known inhibitor) (a), chlorhexidine (a positive control) (b), asiatic acid (c), madecassic acid (d), and madasiatic acid (e) in the active site of GtfC of S. mutans (PDB: 3AIC).Hydrogen bonding interactions were visualized with yellow dotted line, while hydrophobic interactions were represented by gray dotted lines.

Figure 5 :
Figure 5: Inhibition of nitric oxide production in the LPSstimulated RAW264.7 cells treated with the ethanolic C. asiatica extracts and aspirin as a control.* Te statistical signifcance (p value <0.05).
Culture.Te RAW 264.7 cells, a type of mouse macrophage cells, were obtained from ATCC.Te cells were cultured in Dulbecco's modifed Eagle's medium (DMEM; Gibco, Termo Fisher Scientifc, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution ((MTT) solution (Sigma, MO, USA) (0.5 mg/mL in DMEM) was added to each well and incubated for 4 h in the dark after removing the treatment mixture from each well.Te formazan crystals were dissolved by adding 200 μL of dimethylsulfoxide (DMSO) solution (Sigma, MO, USA).Te OD was measured at 570 nm using a microplate reader.Te median lethal concentration (LC50) of substances was calculated by dose-response relationships/sigmoidal curve ftting analysis.Ten percent lethal concentration (LC10) was selected as an appropriate concentration for cellular experiments.Te experiment was performed in triplicate.2.12.Nitric OxideAssays.To assess their potential antiinfammatory efects, the ability of the ethanolic extracts to decrease the production of nitric oxide (NO) induced by lipopolysaccharide (LPS) in RAW 264.7 cells was examined.Te cell suspension was seeded in a 24-well microplate and treated with 72.28 μg/L of the extract with or without 1 μg/ mL of LPS obtained from Sigma-Aldrich (St. Louis, USA).Cells treated with 1 μg/mL of LPS alone were used as a positive control.Aspirin was prepared in DMSO before being added to the well as a negative control.Te fnal concentration of DMSO in the medium was 1/1000 (v/v).2.13.Statistical Analysis.Values of each parameter are expressed as the mean ± standard error of the mean (SEM).Comparisons among diferent groups were performed by one-way analysis of variance (ANOVA).When signifcant diferences existed, Dunnett's multiple-range tests were used to compare the means.A probability of p < 0.05 was considered signifcant.
Extracts of C. asiatica Leaves against Pathogenic Bacteria.Te plant extracts were preliminarily tested for antibacterial activity by agar disk difusion method.Te results are shown in Table1.Te antibacterial activity of the C. asiatica ethanolic extract was noted in the activity of the aqueous extracts.Te ethanolic extract exhibited the greater inhibitory efects against the tested Gram-positive strains than Gram-negative strains in this study.Te inhibition zones of the ethanolic extract against Gram-positive and Gramnegative bacteria were ranging from 9.6-14.3mmand7.3-9.2mm,respectively.Te aqueous extracts showed inhibitory activity against Gram-positive and Gram-negative ranging from 6.5-8.4 mm and 6.2-6.8 mm, respectively.Moreover, the largest inhibitory zone was observed in the efect of C. asiatica ethanolic extract against S. mutans ATCC 25175, which was 14.3 mm.Te disk of vancomycin showed inhibitory activities on Gram-positive bacterial strains ranging from 17.5-24.2mm.Ciprofoxacin provided the efects against Gram-negative strains ranging from 18.5-21.5mm.Te MIC and MBC values of the C. asiatica extracts are shown in Table2.Te MIC and MBC of the ethanolic extracts were better than those of the aqueous extracts.Te ethanolic extracts showed antibacterial activity against Gram-positive bacteria with MICs and MBCs ranging from 1.024 to 2.048 mg/mL and 2.048 to 4.096 mg/mL, respectively, while the aqueous extracts demonstrated MIC values ranging from 16.384 to 32.768 mg/mL, and MBC values were 32.768 to more than 65.536 mg/mL.Te plant ethanolic extract and the aqueous extract possessed the activity to inhibit Gram-negative bacterial strains with the ranging of MIC/MBC values of 8.192 to 16.384/16.384to 32.768 mg/mL and >32.768/>65.536mg/mL, respectively.

Table 1 :
Inhibition zone of the C. asiatica extracts at the concentration of 25 mg per disk and standard antibiotics against pathogenic bacteria on agar culture.
NA: not applicable.

Table 2 :
MICs and MBCs of C. asiatica extracts against pathogenic bacterial strains.
NA: not applicable.

Table 3 :
Te principal compounds isolated from the ethanolic extract of C. asiatica and qualifed by high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA).
3.7.NO Production.NO is a versatile signaling molecule that plays a crucial role in the immune response to infammation.Results of the NO assay (Figure5) established that ethanolic C. asiatica extracts reduced the NO production to 2.66 ± 0.27 M in LPS-stimulated RAW 264.7 cells when compared with untreated LPS-stimulated RAW 264.7 cells (66.15 ± 1.75 μM).Te NO production of aspirin-treated LPS-stimulated RAW 264.7 cells was 7.34 ± 10.44 μM and was not signifcantly diferent from that of the ethanolic C. asiatica extracts treated LPSstimulated RAW 264.7 cells.

Table 4 :
Te binding afnity and interacting amino acid residues of the compounds isolated from C. asiatica ethanolic extract with GtfC from S. mutans (PDB: 3AIC).